DNA Recombination

1. DNA Recombination
• Roles
• Types
• Homologous recombination in E.coli
• Transposable elements

2. Biological Roles for Recombination
1. Generating new gene/allele combinations
(crossing over during meiosis)
2. Generating new genes (e.g., Immuno-
globulin rearrangement)
3. Integration of a specific DNA element (or
virus)
4. DNA repair

3. Practical Uses of Recombination
1. Used to map genes on chromosomes
- recombination frequency proportional
to distance between genes
2. Making transgenic cells and organisms

4. Map of Chromosome I of
Chlamydomonas reinhardtii
Chlamydomonas Genetics Center
cM = centiMorgan; unit of recombination frequency
1 cM = 1% recombination frequency

5. Types of Recombination
1. Homologous - occurs between sequences
that are nearly identical (e.g., during
meiosis)
2. Site-Specific - occurs between sequences
with a limited stretch of similarity; involves
specific sites
3. Transposition – DNA element moves from
one site to another, usually little sequence
similarity involved

6. Examples of (mostly) Homologous Recombination Fig. 22.1

7. Holliday Model
R. Holliday (1964)
- Holliday Junctions
form during
recombination
- HJs can be resolved
2 ways, only one
produces true
recombinant
molecules
patch

8. EM of a Holliday Junction w/a few melted
base pairs around junction
Fig. 22.3

9. Fig. 22.2 a-d
The recBCD
Pathway of
Homologous
Recombination
Part I: Nicking and
Exchanging

10. recBCD Pathway of Homologous Recomb.
Part I: Nicking and Exchanging
1. A nick is created in one strand by recBCD at a Chi
sequence (GCTGGTGG), found every 5000 bp.
2. Unwinding of DNA containing Chi sequence by
recBCD allows binding of SSB and recA.
3. recA promotes strand invasion into homologous
DNA, displacing one strand.
4. The displaced strand base-pairs with the single
strand left behind on the other chromosome.
5. The displaced and now paired strand is nicked
(by recBCD?) to complete strand exchange.

11. recBCD Pathway
of Homologous
Recombination
Part II: Branch
Migration and
Resolution
Fig. 22.5 f-h

12. recBCD Pathway of Homologous Recom.
Part II: Branch Migration and Resolution
1. Nicks are sealed  Holliday Junction
2. Branch migration (ruvA + ruvB)
3. Resolution of Holliday Junction (ruvC)

13. RecBCD : A Complex Enzyme
• RecBCD has:
1. Endonuclease subunits (recBC) that cut
one DNA strand close to Chi sequence.
2. DNA helicase activity (recD subunit) and
a DNA-dependent ATPase activity
– unwinds DNA to generate the 3’ SS tails

14. RecA
• 38 kDa protein that polymerizes onto SS DNA 5’-3’
• Catalyzes strand exchange, also an ATPase
• Also binds DS DNA, but not as strongly as SS

15. RecA binds preferentially
to SS DNA and will
catalyze invasion of a DS
DNA molecule by a SS
homologue.
Important for many types
of homologous
recombination, such as
during meoisis (in yeast).
Fig. 6.19 in Buchanan et al.

16. RecA Function Dissected
• 3 steps of strand exchange:
1. Pre-synapsis: recA coats single-stranded
DNA (accelerated by SSB, so get more
relaxed structure).
2. Synapsis: alignment of complementary
sequences in SS and DS DNA (paranemic
or side-by-side structure).
3. Post-synapsis or strand-exchange: SS DNA
replaces the same strand in the duplex to
form a new DS DNA (requires ATP
hydrolysis).

17. RuvA and RuvB
• DNA helicase that catalyzes branch migration
• RuvA tetramer binds to HJ (each DNA
helix between subunits), forces it into square
planar conformation
• 2 copies of RuvB bind at the HJ (to RuvA and 2
of the DNA helices)
– RuvB is a hexamer ring, has helicase & ATPase
activity
• Branch migration is in the direction of recA
mediated strand-exchange

18. RuvA/RuvB/DNA Complex
Shows RuvB encircling DNA duplexes
RuvB
RuvA

19. Similar to Figure 22.13 Model based on EM images.
RuvB
RuvA
RuvA removed for visual purposes only

20. RuvC : resolvase
• Endonuclease that cuts 2 strands of HJ
• Binds to HJ as a dimer (that already has
RuvA/RuvB)
• Consensus sequence: (A/T)TT (G/C)
- occurs frequently in E. coli genome
- branch migration needed to reach
consensus sequence!

21. RuvC
bound
to a HJ
Fig. 22.16

22. Fig. 22.17b
A model for binding of RuvA, RuvB,
and RuvC to a HJ.

23. Meiotic
Recomb. in
Yeast
- is initiated by a
double-strand
break (DSB)
Fig. 22.18

24. Repair of double-strand breaks (DSBs)
in non-dividing or mitotic cells
DSBs probably most severe form of DNA
damage, can cause loss of genes or even
cell death (apoptosis)
DSBs caused by:
- ionizing radiation
- certain chemicals
- some enzymes (topoisomerases,
endonucleases)
- torsional stress

25. 2 general ways to repair DSBs:
1. Homologous recombination (HR) - repair of broken
DNA using the intact homologue, very similar to
meiotic recombination. Very accurate.
2. Non-homologous end joining (NHEJ) - ligating non-
homologous ends. Prone to errors, ends can be
damaged before religation (genetic material lost) or
get translocations. (Mechanism in Fig 20.38)
Usage: NHEJ >> HR in plants and animals